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Thursday, 26 August 2010

Research paves way for new liver disease research and possibly cell-based therapyThursday, 26 August 2010

By creating diseased liver cells from a small sample of human skin, scientists have for the first time shown that stem cells can be used to model a diverse range of inherited disorders. The University of Cambridge researchers' findings, which will hopefully lead to new treatments for those suffering from liver diseases, were published today in The Journal of Clinical Investigation.

Because liver cells (hepatocytes) cannot be grown in the laboratory, researching liver disorders is extremely difficult. However, today's new research, which was funded by the Wellcome Trust and the Medical Research Council (MRC), demonstrates how to create diseased liver-like cells from patients suffering from a variety of liver disorders.

By replicating the organ's cells, researchers can not only investigate exactly what is happening in a diseased cell, they can also test the effectiveness of new therapies to treat these conditions. It is hoped that their discovery will lead to tailored treatments for specific individuals and eventually cell-based therapy - when cells from patients with genetic diseases are 'cured' and transplanted back. Additionally, as the process could be used to model cells from other parts of the body, their findings could have implications for conditions affecting other organs.

"Our work represents an important step towards delivering the clinical promises of stem cells. However, more work remains to be done and our group is dedicated to achieving this ultimate goal by increasing the knowledge necessary for the development of new therapies."

In the UK, liver disease is the fifth largest cause of death after cardiovascular, cancer, stroke, and respiratory diseases. Over the past 30 years mortality from liver disease in young and middle-aged people has increased over six times, with the number of individuals dying from the disease increasing at a rate of 8-10 per cent every year.

By 2012, the UK is expected to have the highest liver disease death rates in Europe and, without action to tackle the disease, it could overtake stroke and coronary heart disease as the leading cause of death within the next 10-20 years. In the United States, it accounts for approximately 25,000 deaths a year.

For their research, the scientists took skin biopsies from seven patients who suffered from a variety of inherited liver diseases and three healthy individuals (the control group). They then reprogrammed cells from the skin samples back into stem cells. These stem cells were then used to generate liver cells, which mimicked a broad range of liver diseases - the first time patient-specific liver diseases have been modelled using stem cells - and to create 'healthy' liver cells from the control group. Importantly, the three diseases the scientists modelled covered a diverse range of pathological mechanisms, thereby demonstrating the potential application of their research on a wide variety of disorders.

Dr Tamir Rashid of the Laboratory for Regenerative Medicine, University of Cambridge, lead author of the paper, said:

"We know that given the shortage of donor liver organs alternative strategies must urgently be sought. Our study improves the possibility that such alternatives will be found - either using new drugs or a cell-based therapeutic approach."

Professor Mark Thursz, a specialist in liver disease and Professor of Hepatology at Imperial College (who was not affiliated with the study), commented on the importance of the research:

"Liver disease is the fifth most common cause of mortality in many developed countries and unlike the other leading causes of death, the rate of liver related mortality is increasing.”

"The development of patient specific liver cell lines from stem cells is a significant advance in the battle against liver diseases. This technology holds promise in the short term by providing new tools to explore the biology of liver diseases and in the long term as a potential source of liver cells for patients with liver failure."

Human pluripotent stem cells, which can become any other kind of body cell, hold great potential to treat a wide range of ailments, including Parkinson's disease, multiple sclerosis and spinal cord injuries. However, scientists who work with such cells have had trouble growing large enough quantities to perform experiments — in particular, to be used in human studies. Furthermore, most materials now used to grow human stem cells include cells or proteins that come from mice embryos, which help stimulate stem cell growth but would likely cause an immune reaction if injected into a human patient.

To overcome those issues, MIT chemical engineers, materials scientists and biologists have devised a synthetic surface that includes no foreign animal material and allows stem cells to stay alive and continue reproducing themselves for at least three months. It is also the first synthetic material that allows single cells to form colonies of identical cells, which is necessary to identify cells with desired traits and has been difficult to achieve with existing materials.

The research team, led by Professors Robert Langer, Rudolf Jaenisch and Daniel G. Anderson, describes the new material in the Aug. 22 issue of Nature Materials. First authors of the paper are postdoctoral associates Ying Mei and Krishanu Saha.

Human stem cells can come from two sources — embryonic cells or body cells that have been reprogrammed to an immature state. That state, known as pluripotency, allows the cells to develop into any kind of specialized body cells.

It also allows the possibility of treating nearly any kind of disease that involves injuries to cells. Scientists could grow new neurons for patients with spinal cord injuries, for example, or new insulin-producing cells for people with type 1 diabetes.

To engineer such treatments, scientists would need to be able to grow stem cells in the lab for an extended period of time, manipulate their genes, and grow colonies of identical cells after they have been genetically modified. Current growth surfaces, consisting of a plastic dish coated with a layer of gelatine and then a layer of mouse cells or proteins, are notoriously inefficient, says Saha, who works in Jaenisch's lab at the Whitehead Institute for Biomedical Research.

"If we can make it easier for the cells to divide and grow, that will really help to get the number of cells you need to do all of the disease studies that people are excited about."

Vitronectin.

Previous studies had suggested that several chemical and physical properties of surfaces — including roughness, stiffness and affinity for water — might play a role in stem cell growth. The researchers created about 500 polymers (long chains of repeating molecules) that varied in those traits, grew stem cells on them and analyzed each polymer's performance. After correlating surface characteristics with performance, they found that there was an optimal range of surface hydrophobicity (water-repelling behaviour), but varying roughness and stiffness did not have much effect on cell growth.

They also adjusted the composition of the materials, including proteins embedded in the polymer. They found that the best polymers contained a high percentage of acrylates, a common ingredient in plastics, and were coated with a protein called vitronectin, which encourages cells to attach to surfaces.

Using their best-performing material, the researchers got stem cells (both embryonic and induced pluripotent) to continue growing and dividing for up to three months. They were also able to generate large quantities of cells — in the millions.

The MIT researchers hope to refine their knowledge to help them build materials suited to other types of cells, says Anderson, from the MIT Department of Chemical Engineering, the Harvard-MIT Division of Health Sciences and Technology, and the David H. Koch Institute for Integrative Cancer Research.

"We want to better understand the interactions between the cell, the surface and the proteins, and define more clearly what it takes to get the cells to grow," he says.

Friday, 20 August 2010

Physical model describes the distribution of nucleosomesFriday, 20 August 2010

The crystal structure of the nucleosome

core particle consisting of H2A, H2B,

H3 and H4 and DNA. The view is from

the top through the superhelical axis.

The DNA genomes of organisms whose cells possess nuclei are packaged in a highly characteristic fashion. Most of the DNA is tightly wrapped around protein particles called nucleosomes, which are connected to each other by flexible DNA segments, like pearls on a necklace. This arrangement plays a major role in deciding which genes are actively expressed, and thus which proteins can be synthesized in a given cell.

The biophysicists Professor Ulrich Gerland and Wolfram Moebius from Ludwig-Maximilians-Universitaet (LMU) in Munich have recently developed a model which explains the distribution of nucleosomes around the functionally crucial transcription start sites. Transcription is the first step in the process that converts genetic information into proteins. At the transcription start sites the DNA must be free of nucleosomes. The two researchers discovered that distinct stop signals positioned on either side of these zones must actively prevent the formation and sliding of nucleosomes.

"Our model provides a useful tool for dissecting the so-called chromatin code, which determines how the DNA is packed and selectively made accessible for transcription", says Gerland.

In higher organisms, the genetic material in each cell is packed in the form of compact chromosomes in the nucleus. The basic structural unit of a chromosome is the nucleosome. The nucleosomes, each made up of two copies of four different histone proteins, provide a kind of spool on which the DNA strands are wound, and are linked together by more flexible sections of DNA, like beads on a string. But nucleosomes are not just passive packages that keep the DNA in a compact form.

"They have a decisive influence on gene regulation, insofar as they help to control which segments of the DNA can be translated into proteins", explains Gerland, Arnold Sommerfeld Center for Theoretical Physics (ASC) and Center for NanoScience (CeNS) in the Physics Faculty of LMU Munich.

The accessibility of the DNA is a primary determinant of gene expression, and is therefore of great interest to molecular geneticists. A central question is how nucleosomes are distributed around the regions at which transcription starts. The selection of the start site, or gene promoter, is the first crucial step in the conversion of genetic information into the bricks and mortar of all cells, the proteins. It turns out that these promoter sites are marked by the presence of a nucleosome-free zone flanked by a specific pattern of nucleosomes. The biological function of these gaps seems to be to provide accessible docking sites for the transcriptional machinery, which comprises a multi-protein complex consisting of many subunits.

Together with his PhD student Wolfram Moebius, Gerland has asked whether a simple physical principle might not account for the characteristic distribution of nucleosomes in the vicinity of transcription start sites. The researchers made use of the so-called Tonks model, which applies to interactions between diffusing particles that are confined to one dimension.

"Provided one knows the position of a single particle, one can use the model to predict, in a statistical sense, the positions of the particles in the vicinity", says Wolfram Moebius, who is the first author on the new study.

"In addition, one observes a typical pattern of oscillations in the particle density."

The analyses showed that the model of a Tonks gas indeed describes the distribution of the nucleosomes with surprising precision.

"When we plug average values derived from a large set of promoter regions into the model, the calculations reproduce the typical range of variation in nucleosome density that we see in biological systems", explains Gerland. The new model agrees best with the biological data if one assumes that the boundaries on either side of a nucleosome-free zone are defined by different conditions.

"On the side nearer the transcription initiation site, there must be a fixed nucleosome that prevents sliding along the DNA, like a sign saying 'Road Closed'", explains Gerland.

"At the other end of the open stretch there has to be a larger segment that is refractory to nucleosome assembly. In other words, there must be a signal that serves as a No Parking sign for nucleosomes."

The results obtained by Moebius and Gerland for the first time quantitatively confirm a statistical model for the distribution of nucleosomes in the genome proposed by the American biochemist Roger Kornberg, (who first discovered nucleosomes in 1974, and won the Nobel Prize for Medicine for his studies on the structure of RNA polymerase in 2009). The new model contributes to our understanding of the rules that determine how chromosome structure is established and modulated.

"Our calculations should certainly help to decode the so-called chromatin code, the basis for which is not well understood", says Gerland.

"This code provides the blueprint for the three-dimensional structure of the genome."

The first successful report of using cell-depleted lung as a natural growth matrix for generating new rat lung from embryonic stem cells is presented in a breakthrough article in Tissue Engineering, Part A, a peer-reviewed journal published by Mary Ann Liebert, Inc..

Embryonic stem cells (ESCs) have the potential to mature into virtually any type of cell and tissue type, but they require an appropriate environment and chemical signals to drive their differentiation into specific cell types and to form 3-dimensional tissue structures. Alternatives to available synthetic tissue matrices are needed to drive this technology forward and develop clinical applications for engineered lung tissue.

Joaquin Cortiella, MD, MPH, and colleagues from University of Texas Medical Branch (Galveston), Stanford University (Palo Alto, CA), Brown Medical School (Providence, RI), and Duke University (Durham, NC), describe the first attempt to make acellular rat lung and use it as a biological matrix for differentiating ESCs into lung tissue. The authors present evidence of improved cell retention, repopulation of the matrix, and differentiation into the cell types present in healthy lung. They also report signs that the cells are organizing into the 3-D structures characteristic of complex tissues and are producing the chemical signals and growth factors that guide lung tissue function and development.

Cortiella and co-authors describe the process used to remove the cellular component of natural lung tissue and create a growth matrix for ESCs in the article: "Influence of Acellular Natural Lung Matrix on Murine Embryonic Stem Cell Differentiation and Tissue Formation".

"Organ-specific extracellular matrices, properly prepared, are serving more and more as the appropriate structural scaffold for the recapitulation of a specific organ's tissues. This turns out to be especially true in an organ such as the lung, whose parenchyma must have a structure that accommodates atmospheric gas transmission as well as vascular, lymphatic, and neural systems," says Peter C. Johnson, MD, Co-Editor-in-Chief of Tissue Engineering and Vice President, Research and Development, Avery Dennison Medical Products.

UCI study is first to show reversal of long-term hind-limb paralysis Friday, 20 August 2010

A UC Irvine study is the first to demonstrate that human neural stem cells can restore mobility in cases of chronic spinal cord injury, suggesting the prospect of treating a much broader population of patients.

Human neural stem cells transplanted

into mice grew into neural tissue cells,

such as oligodendrocytes.

Credit: Brian Cummings - UCI.

Previous breakthrough stem cell studies have focused on the acute, or early, phase of spinal cord injury, a period of up to a few weeks after the initial trauma when drug treatments can lead to some functional recovery.

The UCI study, led by Aileen Anderson and Brian Cummings of the Sue & Bill Gross Stem Cell Research Center, is significant because the therapy can restore mobility during the later chronic phase, the period after spinal cord injury in which inflammation has stabilized and recovery has reached a plateau. There are no drug treatments to help restore function in such cases.

The study appears in the open-access, peer-reviewed journal PLoS ONE.

The Anderson-Cummings team transplanted human neural stem cells into mice 30 days after a spinal cord injury caused hind-limb paralysis. The cells then differentiated into neural tissue cells, such as oligodendrocytes and early neurons, and migrated to spinal cord injury sites. Three months after initial treatment, the mice demonstrated significant and persistent recovery of walking ability in two separate tests of motor function when compared to control groups.

The research is the latest in a series of collaborative studies conducted since 2002 with StemCells Inc. that have focused on the use of StemCells' human neural stem cells in spinal cord injury and resulted in multiple co-authored publications. StemCells Inc., based in Palo Alto, Calif., is engaged in the research, development and commercialization of stem cell therapeutics and tools for use in stem cell-based research and drug discovery.

According to Dr. Stephen Huhn, vice president and head of the central nervous system program at StemCells Inc., "the strong preclinical data we have accumulated to date will enable our transition to a clinical trial, which we plan to initiate in 2011."

Tuesday, 17 August 2010

Rice statisticians confirm date of 'mitochondrial Eve' with new methodTuesday, 17 August 2010

The most robust statistical examination to date of our species' genetic links to "mitochondrial Eve" — the maternal ancestor of all living humans confirms that she lived about 200,000 years ago. The Rice University study was based on a side-by-side comparison of 10 human genetic models that each aim to determine when Eve lived using a very different set of assumptions about the way humans migrated, expanded and spread across Earth.

The research is available online in the journal Theoretical Population Biology.

"Our findings underscore the importance of taking into account the random nature of population processes like growth and extinction," said study co-author Marek Kimmel, professor of statistics at Rice.

"Classical, deterministic models, including several that have previously been applied to the dating of mitochondrial Eve, do not fully account for these random processes."

The quest to date mitochondrial Eve (mtEve) is an example of the way scientists probe the genetic past to learn more about mutation, selection and other genetic processes that play key roles in disease.

"This is why we are interested in patterns of genetic variability in general," Kimmel said.

"They are very important for medicine."

For example, the way scientists attempt to date mtEve relies on modern genetic techniques. Genetic profiles of random blood donors are compared, and based upon the likenesses and differences between particular genes, scientists can assign a number that describes the degree to which any two donors are related to one another.

Using mitochondrial genomes to gauge relatedness is a way for geneticists to simplify the task of finding common ancestors that lived long ago. That is because the entire human genome contains more than 20,000 genes, and comparing the differences among so many genes for distant relatives is problematic, even with today's largest and fastest supercomputers.

But mitochondria — the tiny organelles that serve as energy factories inside all human cells — have their own genome. Besides containing 37 genes that rarely change, they contain a "hypervariable" region, which changes fast enough to provide a molecular clock calibrated to times comparable to the age of modern humanity. Because each person's mitochondrial genome is inherited from his or her mother, all mitochondrial lineages are maternal.

To infer mtEve's age, scientists must convert the measures of relatedness between random blood donors into a measure of time.

"You have to translate the differences between gene sequences into how they evolved in time," said co-author Krzysztof Cyran, vice head of the Institute of Informatics at Silesian University of Technology in Gliwice, Poland.

"And how they evolved in time depends upon the model of evolution that you use. So, for instance, what is the rate of genetic mutation, and is that rate of change uniform in time? And what about the process of random loss of genetic variants, which we call genetic drift?"

Within each model, the answers to these questions take the form of coefficients — numeric constants that are plugged into the equation that returns the answer for when mtEve lived.

Each model has its own assumptions, and each assumption has mathematical implications. To further complicate matters, some of the assumptions are not valid for human populations. For example, some models assume that population size never changes. That is not true for humans, whose population has grown exponentially for at least several thousand generations. Other models assume perfect mixing of genes, meaning that any two humans anywhere in the world have an equal chance of producing offspring.

Cyran said human genetic models have become more complex over the past couple of decades as theorists have tried to correct for invalid assumptions. But some of the corrections — like adding branching processes that attempt to capture the dynamics of population growth in early human migrations — are extremely complex. Which raises the question of whether less complex models might do equally well in capturing what is occurring.

"We wanted to see how sensitive the estimates were to the assumptions of the models," Kimmel said.

"We found that all of the models that accounted for random population size — such as different branching processes — gave similar estimates. This is reassuring, because it shows that refining the assumptions of the model, beyond a certain point, may not be that important in the big picture."

A study led by a researcher at Albert Einstein College of Medicine of Yeshiva University has revealed a unique "partnership" between two types of bone marrow stem cells, which could lead to advances in regenerative medicine. The aim of regenerative medicine is to enable the body to repair, replace, restore or regenerate damaged or diseased cells, tissues and organs.

Hematopoietic stem cells (HSCs) in the bone marrow perform the vital task of producing all blood cells in the human body. Now, the new study, published in the August 12 issue of Nature, has revealed that HSCs pair up in the bone marrow with another type of stem cells, known as mesenchymal stem cells, which give rise to bone, cartilage, fat and other tissues. This pairing, the research shows, form a unique stem cell "partnership" that could lead to advances in regenerative medicine.

The identity of cells in close proximity to HSCs had been a matter of dispute. Dr. Frenette and his team not only found that HSCs and mesenchymal stem cells partner physically with each other, but they also showed that the two types of stem cells interact in crucially important ways. Mesenchymal cells, for example, were found to be necessary for keeping HSCs in the bone marrow alive.

"We think that these mesenchymal stem cells are a very important component of the stem cell niche in the bone marrow," said Dr. Frenette.

"These cells likely play important roles in stem cell maintenance, movement, and regeneration of the bone marrow. Further studies into their functions might allow us to maintain healthy stem cells and develop new methods to expand them for clinical use."

Monday, 16 August 2010

Technologies developed at the Buck Institute can speed the manufacturing of authentic neurons from stem cells for future clinical applicationsMonday, 16 August 2010

Researchers at the Buck Institute for Age Research have successfully used human induced pluripotent stem cells (iPSCs) to treat rodents afflicted with Parkinson's Disease (PD). The research, which validates a scalable protocol that the same group had previously developed, can be used to manufacture the type of neurons needed to treat the disease and paves the way for the use of iPSC's in various biomedical applications. Results of the research, from the laboratory of Buck faculty Xianmin Zeng, Ph.D., are published August 16, 2010 in the on-line edition of the journal Stem Cells.

Human iPSC's are a "hot" topic among scientists focused on regenerative medicine.

"These cells are reprogrammed from existing cells and represent a promising unlimited source for generating patient-specific cells for biomedical research and personalized medicine," said Zeng, who is lead author of the study.

"Human iPSCs may provide an end-run around immune-rejection issues surrounding the use of human embryonic stem cells (hESCs) to treat disease," said Zeng.

"They may also solve bioethical issues surrounding hESCs."

Researchers in the Zeng lab used human iPSCs that were derived from skin and blood cells and coaxed them to become dopamine-producing neurons. Dopamine is a neurotransmitter produced in the mid-brain, which facilitates many critical functions, including motor skills. Patients with PD lack sufficient dopamine; the disease is a progressive, incurable neurodegenerative disorder that affects 1.5 million Americans and results in tremor, slowness of movement and rigidity.

Researchers transplanted the iPSC-derived neurons into rats that had mid-brain injury similar to that found in human PD. The cells became functional and the rats showed improvement in their motor skills. Zeng said this is the first time iPSC-derived cells have been shown to engraft and ameliorate behavioural deficits in animals with PD. Dopamine-producing neurons derived from hESCs have been demonstrated to survive and correct behavioural deficits in PD in the past.

"Both our functional studies and genomic analyses suggest that overall iPSCs are largely similar to hESCs," said Zeng.

The research also addresses the current lack of a robust system for the efficient production of functional dopamine-producing neurons from human iPSCs, Zeng said. The protocol used to differentiate the iPSCs was similar to one developed by Zeng and colleagues for hESCs.

"Our approach will facilitate the adoption of protocols to good manufacturing practice standards, which is a pre-requisite if we are to move iPSC's into clinical trials in humans," said Zeng.

"The researchers showed they could produce quantities of dopaminergic neurons necessary to improve the behaviour of a rodent model of PD. We look forward to further work that could bring closer a new treatment for such a debilitating disease," Trounson said.

Having charted the occurrence of a common chemical change that takes place while stem cells decide their fates and progress from precursor to progeny, a Johns Hopkins-led team of scientists has produced the first-ever epigenetic landscape map for tissue differentiation.

The details of this collaborative study between Johns Hopkins, Stanford and Harvard appear August 15 in the early online publication of Nature.

The researchers, using blood-forming stem cells from mice, focused their investigation specifically on an epigenetic mark known as methylation. This change is found in one of the building blocks of DNA, is remembered by a cell when it divides, and often is associated with turning off genes.

Employing a customized genome-wide methylation-profiling method dubbed CHARM (comprehensive high-throughput arrays for relative methylation), the team analyzed 4.6 million potentially methylated sites in a variety of blood cells from mice to see where DNA methylation changes occurred during the normal differentiation process. The team chose the blood cell system as its model because it's well-understood in terms of cellular development.

They looked at eight types of cells in various stages of commitment, including very early blood stem cells that had yet to differentiate into red and white blood cells. They also looked at cells that are more committed to differentiation: the precursors of the two major types of white blood cells, lymphocytes and myeloid cells. Finally, they looked at older cells that were close to their ultimate fates to get more complete pictures of the precursor-progeny relationships — for example, at white blood cells that had gone fairly far in T-cell lymphocyte development. (Lymphoid and myeloid constitute the two major types of progenitor blood cells.)

"It wasn't a complete tree, but it was large portions of the tree, and different branches," says Andrew Feinberg, M.D., M.P.H., King Fahd Professor of Molecular Medicine and director of the Center for Epigenetics at Hopkins' Institute for Basic Biomedical Sciences.

"Genes themselves aren't going to tell us what's really responsible for the great diversity in cell types in a complex organism like ourselves," Feinberg says.

"But I think epigenetics — and how it controls genes — can. That's why we wanted to know what was happening generally to the levels of DNA methylation as cells differentiate."

One of the surprising finds was how widely DNA methylation patterns vary in cells as they differentiate.

"It wasn't a boring linear process," Feinberg says.

"Instead, we saw these waves of change during the development of these cell types."

The data shows that when all is said and done, the lymphocytes had many more methylated genes than myeloid cells. However, on the way to becoming highly methylated, lymphocytes experience a huge wave of loss of DNA methylation early in development and then a regain of methylation. The myeloid cells, on the other hand, undergo a wave of increased methylation early in development and then erase that methylation later in development.

Rudimentary as it is, this first epigenetic landscape map has predictive power in the reverse direction, according to Feinberg. The team could tell which types of stem cells the blood cells had come from, because epigenetically those blood cells had not fully let go of their past; they had residual marks that were characteristic of their lineage.

This project involved a repertoire of talents.

"None of whom were more integral than Irv Weissman at Stanford," Feinberg says.

"He's a great stem cell biologist and he lent a whole level of expertise that we didn't have."

One apparent application of this work might be to employ these same techniques to assess how completely an induced pluripotent stem cell (iPSC) has been reprogrammed.

"You might want to have an incompletely reprogrammed cell type from blood, for example, that you take just to a certain point because then you want to turn it into a different kind of blood cell," Feinberg says, cautioning that the various applications are strictly theoretical.

Because the data seem to indicate discreet stages of cell differentiation characterized by waves of changes in one direction and subsequent waves in another, cell types conceivably could be redefined according to epigenetic marks that will provide new insights into both normal development and disease processes.

Discovery furthers knowledge of microRNA's role in diseaseMonday, 16 August 2010

Researchers at Tufts University School of Medicine and Tufts Medical Center have identified an RNA sequence that promotes increased numbers of specific microRNAs (miRNAs), molecules that regulate cell growth, development, and stress response. The discovery helps researchers understand the links between miRNA expression and disease, including heart disease and cancer. The findings are published in the August 13 issue of Molecular Cell.

"A growing body of evidence shows that abnormal expression of miRNAs can contribute to human diseases such as heart disease and cancer. A better understanding of how miRNAs are generated and how they regulate genes may provide important insights into the mechanisms of physiological disorders such as heart disease and cancer," said senior author Akiko Hata, PhD, associate professor in the department of biochemistry at Tufts University School of Medicine (TUSM) and a member of the biochemistry and cell, molecular and developmental biology program faculties at the Sackler School of Graduate Biomedical Sciences at Tufts.

MiRNAs are initially formed as a long sequence of RNA called the primary miRNA. This molecule undergoes several steps to transform it into mature miRNA. Once formed, the mature miRNAs regulate gene expression by silencing or activating target genes. More than 700 human miRNAs with various functions are currently known.

Hata and colleagues previously found that the processing of some miRNAs could be regulated in response to cellular signals from a specific signalling pathway. In the current study, Hata and colleagues found that most of the miRNAs regulated by this signalling pathway share a common RNA sequence. When this RNA sequence was mutated, the signalling pathway no longer regulated miRNA processing. Conversely, when the RNA sequence was introduced into a new miRNA, the miRNA became responsive to the signalling pathway.

"An enzyme called Drosha is needed for miRNA processing. Our previous studies determined that proteins called Smads are also required for the processing of some miRNAs in response to cellular signals. Now, we have identified the RNA sequence that recruits Drosha and Smads for miRNA processing in response to the signalling pathway," said first author Brandi Davis, PhD, a 2010 graduate of the biochemistry program at the Sackler School and a postdoctoral fellow in Hata's lab.

"We knew that Smad proteins regulate gene expression by binding to DNA. Our current study is exciting because it shows that Smads play an additional role, controlling miRNA expression by binding to the structurally different RNA."

While miRNAs were first discovered in 1993, scientists did not link them to gene regulation until nearly ten years later. Now, scientists are working to understand how miRNA expression is controlled, what genes miRNAs target, and how varying levels of miRNAs are related to human disease, particularly heart disease and cancer.

"Scientists are just beginning to understand the roles of miRNA in the body, and this study adds another piece to the puzzle. By investigating the mechanisms that govern which genes are translated and which genes are silenced, we can begin to understand how miRNAs impact the progression of cardiovascular diseases and cancer," said Hata.

Hata is also the director of the Molecular Signaling Laboratory in the Molecular Cardiology Research Institute (MCRI) at Tufts Medical Center. The MCRI, with investigators and physician-scientists from Tufts University School of Medicine and Tufts Medical Center, is dedicated to the study of the molecular mechanisms of human cardiovascular disease, the translation of bench findings to new bedside strategies for diagnosis and therapy, and the mentoring of MD and PhD trainees committed to a career in academic cardiovascular research.

Thursday, 12 August 2010

Stem cells used to treat children with recessive dystrophic epidermolysis bullosaThursday, 12 August 2010

University of Minnesota Physician-researchers have demonstrated that a lethal skin disease can be successfully treated with stem cell therapy.

Medical School researchers John E. Wagner, M.D., and Jakub Tolar, M.D., Ph.D., in collaboration with researchers in Portland, Oregon, the United Kingdom, and Japan have for the first time used stem cells from bone marrow to repair the skin of patients with a fatal skin disease called recessive dystrophic epidermolysis bullosa, or RDEB. This is the first time researchers have shown that bone marrow stem cells can home to the skin and upper gastrointestinal tract and alter the natural course of the disease.

"Whether stem cells from marrow could repair tissues other than itself has been quite controversial," said Wagner, director of paediatric blood and marrow transplantation and clinical director of the Stem Cell Institute.

"But in 2007 we found a rare subpopulation of marrow stem cells that could repair the skin in laboratory models. This astounding finding compelled us to test these stem cells in humans. This has never been done before."

"This discovery is more unique and more remarkable than it may first sound because until now, bone marrow has only been used to replace diseased or damaged marrow – which makes sense," said Tolar, associate professor of paediatric transplantation.

"But what we have found is that stem cells contained in bone marrow can travel to sites of injured skin, leading to increased production of collagen which is deficient in patients with RDEB."

Epidermolysis bullosa (EB), is a rare, genetic skin disease that causes skin to blister and scrape off with the slightest friction or trauma. It affects the skin and lining of the mouth and oesophagus. Previously, there was no treatment and no chance for cure. In some countries, even euthanasia has been considered for newborns with the severest forms. If children with EB do not die of infection in their early life, many with the disease do not live beyond their 20s or 30s because they develop an aggressive form of skin cancer. While a few will live long term, the severest forms of EB are generally lethal.

"Bone marrow transplantation is one of the riskiest procedures in medicine, yet it is also one of the most successful," said Tolar.

"Patients who otherwise would have died from their disease can often now be cured. It's a serious treatment for a serious disease."

Wagner and Tolar initiated the study in the fall of 2007. Since then, 10 children with the most aggressive forms of EB have been transplanted at the University of Minnesota Amplatz Children's Hospital. While all of the children have responded to the therapy, the magnitude of each response has varied.

"To understand this achievement, you have to understand how horrible this disease actually is," said Wagner.

"From the moment of birth, these children develop blisters from the slightest trauma which eventually scar. They live lives of chronic pain, preventing any chance for a normal life. My hope is to do something that might change the natural history of this disease and enhance the quality of life of these kids."

Wagner and Tolar are measuring the progress each child makes after treatment in several ways. Clinically, the physicians monitor any improvements in health as well as in the strength of the recipient's skin after transplant. They also use laboratory measures to determine how well the donor's cells are engrafting in – or becoming an integral part of – the skin, as well as measure the levels of collagen 7 – the protein missing in children with RDEB which is responsible for keeping layers of skin 'glued' to one another and to the body.

"What we now know is that after this treatment, healthy donor cells reside in the skin, collagen 7 consistently increases over time and the skin gradually becomes more resistant to blister formation." said Wagner.

"This discovery expands the scope of marrow transplantation and serves as an example of the power of stem cells in the treatment of disease."

"While the treatment offers a chance for a better life, it comes with significant risk," said Tolar.

"Two children have died from complications related to the treatment, so refinements are needed."

In fact, earlier this year Wagner and Tolar launched a new generation of the study by combining different stem cell populations.

"We are fully conscious of what we have accomplished so far and the enormity of what else needs to be done," said Tolar.

"But we have one goal — to take EB off the incurable list."

This breakthrough is another example of the University of Minnesota Medical School's continued excellence in regenerative medicine therapies. The world's first successful human bone marrow transplant was performed at the University in 1968, and since then, University of Minnesota Physicians have achieved many other world-firsts. Others include the first successful transplant to treat lymphoma (1975), the first use of umbilical cord blood to treat leukaemia (1990), the first use of embryo selection to prevent a genetic disease and guarantee a human leukocyte antigen (HLA)-matched sibling ('saviour sibling') and the first use of multi-unit umbilical cord blood transplantation to treat adults (2000).

Researchers are optimistic their discovery will translate into therapy critically ill patients on the verge of respiratory failureThursday, 12 August 2010

Researchers are reporting this week new study results they say provide further evidence of the therapeutic potential of stem cells derived from bone marrow for patients suffering from acute lung injury, one of the most common causes of respiratory failure in intensive care units.

Led by Drs. Michael A. Matthay and Jae W. Lee at the Cardiovascular Research Institute of the University of California, San Francisco, the team writes in a Journal of Biological Chemistry "Paper of the Week" that its experiments have revealed how a type of bone marrow stem cell bolsters damaged lung cells.

"We found that these stem cells secreted a significant quantity of a protein that restored the barrier that keeps fluid and other elements out of the lungs," said Lee, an associate professor of anaesthesia at UCSF.

"We're optimistic about the promise that future clinical trials may hold."

Scientists for decades have harnessed the natural regenerative properties of bone marrow to treat patients with blood-related diseases. And, of late, investigations into the potential of using bone marrow stem cells to treat damaged tissues have intensified.

There are two types of stem cells in bone marrow. One kind, hematopoietic stem cells, is tasked with producing red and white blood cells, depending upon the immune system's needs. The other, mesenchymal stem cells, is the focus of Matthay and Lee's work. While mesenchymal stem cells also support the production of blood cells, scientists today are quite interested in their ability to differentiate into cells that, when mature, develop into tissues throughout the body.

"Within the past several years, there has been an increased interest in understanding the biology of stem cells for clinical use as cell-based therapies," Lee said.

A number of conditions, such as pneumonia and sepsis, also known as blood poisoning, brings on acute lung injury. In some cases, acute lung injury develops into a more serious condition, known as acute respiratory distress syndrome, and results in insufficient oxygenation of blood and eventual organ failure.

Buried in the depths of healthy lung tissue, tiny groups of cells called alveoli stretch open to accommodate oxygen with each breath and then remove carbon dioxide during exhalation. Each alveolus is lined with a layer of epithelial cells that serve as a critical barrier – keeping certain substances in and certain substances out – so that the gas balance inside is appropriately maintained.

In contrast, inflammation due to injury or infection can make the border of epithelial cells become more porous than it should be. The increased permeability allows an often-deadly mix of substances, such as fluid and cells, to seep into and accumulate in the alveoli.

Despite extensive research on acute lung injury and acute respiratory distress syndrome, the mortality rate for patients remains high – at about 40 percent, Lee said, and pharmacological therapies that reduce the severity of lung injury in experimental studies have not yet translated into effective clinical treatment options.

The team decided to re-create the unhealthy lung conditions in the lab – by culturing human alveolar cells and then chemically causing inflammation – and to observe how the presence of bone marrow stem cells would change things.

"We then introduced mesenchymal stem cells without direct cell contact, and they churned out a lot of protein, called angiopoietin-1, which prevented the increase in lung epithelial permeability after the inflammatory injury," said Xiaohui Fang, the first author of the manuscript.

The authors say the findings are the first to demonstrate how mesenchymal stem cells revive the epithelial border of the alveoli, and they hope clinical trials will prove the therapy is a viable one for preventing respiratory failure in critically ill patients.

Transposons: DNA that May Contribute to Each Person's UniquenessThursday, 12 August 2010

Building on a tool that they developed in yeast four years ago, researchers at the Johns Hopkins University School of Medicine scanned the human genome and discovered what they believe is the reason people have such a variety of physical traits and disease risks.

In a report published in the June 25 issue of Cell, the team identified a near complete catalogue of the DNA segments that copy themselves, move around in, and insert themselves here and there in our genome. The insertion locations of these moveable segments — transposons — in each individual's genome helps determine why some are short or tall, blond or brunette, and more likely or less likely to have cancer or heart disease. The Johns Hopkins researchers say that tracking the locations of transposons in people with specific diseases might lead to the discovery of new disease genes or mutations.

Using their specialized "chip" with DNA spots that contain all of the DNA sequences that appear in the genome, researchers applied human DNA from 15 unrelated people. The research team compared transposon sites first identified in the original published human "index" genome and found approximately 100 new transposon sites in each person screened.

"We were surprised by how many novel insertions we were able to find," says Jef Boeke, Ph.D., Sc.D., an author on the article, a professor of molecular biology and genetics, and co-director of the High Throughput Biology Center of the Institute for Basic Biomedical Sciences at Johns Hopkins.

"A single microarray experiment was able to reveal such a large number of new insertions that no one had ever reported before. The discovery taught us that these transposons are much more active than we had guessed."

Each of the 15 different DNA samples used in the study was purified from blood cells before it was applied to a DNA chip. Transposons stick to spots on the DNA chip corresponding to where they are normally found in the genome, letting the researchers locate new ones.

Boeke's group first invented the transposon chip in 2006 for use in yeast. However, it was Kathleen Burns, M.D., Ph.D., now an assistant professor of pathology at Johns Hopkins, who first got the chip to work with human DNA.

"The human genome is much larger and more complex, and there are lots of look-a-like DNA’s that are not actively moving but are similar to the transposons that we were interested in," says Burns.

The trick was to modify how they copied the DNA before it was applied over the chip. The team was able to copy DNA from the transposons of interest, which have just three different genetic code letters than other look-alike DNA segments.

"We've known that genomes aren't static places, but we didn't know how many transposons there are in each one of us; we didn't know how often a child is born with a new one that isn't found in either parent and we didn't know if these DNA’s were moving around in diseases like cancer," says Burns.

"Now we have a tool for answering these questions. This adds a whole dimension to how we look at our DNA."

Tuesday, 10 August 2010

These days people usually don't die from a heart attack. But the damage to heart muscle is irreversible, and most patients eventually succumb to congestive heart failure, the most common cause of death in developed countries.

Stem cells now offer hope for achieving what the body cannot do: mending broken hearts. Engineers and physicians at the University of Washington have built a scaffold that supports the growth and integration of stem cell-derived cardiac muscle cells. A description of the scaffold, which supports the growth of cardiac cells in the lab and encourages blood vessel growth in living animals, is published this week in the Proceedings of the National Academy of Sciences.

"Today, if you have a heart attack there's nothing that doctors can do to repair the damage," said lead author Buddy Ratner, a UW professor of bioengineering.

"Your body can't make new heart cells, but what if we can deliver vital new cells in that damaged portion of the heart?"

Experiments in a culture dish show

that chick heart cells (red) grow in the

scaffold channels (green) at densities
similar to those in a living heart.

Credit: University of Washington.

Ratner and his colleagues built a tiny tubular porous scaffold that supports and stabilizes the fragile cardiac cells and can be injected into a damaged heart, where it will foster cell growth and eventually dissolve away. The new scaffold not only supports cardiac muscle growth, but potentially accelerates the body's ability to supply oxygen and nutrients to the transplanted tissue. Eventually, the idea is that doctors would seed the scaffold with stem cells from either the patient or a donor, then implant it when the patient is treated for a heart attack, before scar tissue has formed.

Other heart scaffolds or tissue patches currently being developed combine cardiac muscle cells and two other types of cells needed to kick-start the growth of blood vessels and connective tissue. Preparing each type of cells is an enormous amount of work, so a scaffold that requires just one type of cell, like this one, would be significantly cheaper and easier to use.

Ratner's scaffold is a flexible polymer with interconnected pores all of the same size. This one also includes channels to accommodate cardiac cells' preference for fusing together in long chains. Researchers first verified the design using chicken embryonic heart cells, and confirmed that the scaffold could support heart tissue growth at concentrations similar to those in living heart tissue.

They then seeded the scaffold with cardiac muscle cells derived from human embryonic stem cells. These cells survived and collected in the channels. Over five days, the cardiac muscle cells multiplied faster in the scaffold environment than other cell types, and could survive up to 300 micrometers (about the diameter of four human hairs) from the scaffold edge – an important point if the scaffold is to integrate with the body.

The cells expressed two proteins associated with muscle contraction and could contract with sufficient force to deform the scaffold.

The scaffold is built out of a flexible,

biocompatible material with

30-micrometer pores that support the

fragile cardiac cells and allow access

to blood and nutrients. The scaffold

for heart repair includes 60-micrometer

channels, seen here as the larger holes,

where the cardiac cells can fuse into

long chains. Credit: University of

Washington.

Researchers also implanted a bare scaffold into a living rat's heart to verify the scaffold's biocompatibility. Results showed that after four weeks, the heart had accepted the foreign body, and new blood vessels had penetrated into the scaffold.

Why blood vessels penetrate so well is unknown. One hypothesis involves the macrophage, a cell in the immune system, and the size of the pores, which seems to be critical. The macrophages first attack the foreign body as an invader and try to digest it. They enter the pores and are themselves entrapped. At this point, the macrophage seems to switch from its attack mode to its healing mode. The team is now investigating the blood vessel formation.

Heart tissues need a rich blood supply, and that has been one of the limiting factors to heart repair and vascular tissue engineering, said co-author Chuck Murry, professor of pathology and bioengineering.

"The first thing that transplanted heart cells have to do is survive. And when you transition them from a culture dish to the body, initially they don't have a blood supply. So we have to promote the host blood supply as fast as possible," Murry said.

"We're very optimistic that this scaffold will help keep the muscle cells alive after implantation and will help transition them to working heart muscles," Murry said.

The scaffold is made from a jelly-like hydrogel material developed by first author, UW bioengineering doctoral student Lauran Madden. A needle is used to implant the tiny (third of a millimetre wide by 4 millimetres long) scaffold rods into the heart muscle. But the scaffold can support growth of larger clumps of heart tissue, Madden said.

The next steps will involve adjusting the scaffold degradation time so that the scaffold degrades at the same rate that cardiac cells proliferate and that blood vessels and support fibres grow in, and then implant a cell-laden scaffold into a damaged heart.

"What we have now is a really good system going in the dish, and we're working to transition it to in the body," Madden said.

Beat BioTherapeutics, a Seattle start-up co-founded by Ratner, Murry and co-author Michael Laflamme, a UW assistant professor of pathology, plans to license the technology to help bring it to patients.

Monday, 9 August 2010

Researchers for the first time have induced robust regeneration of nerve connections that control voluntary movement after spinal cord injury, showing the potential for new therapeutic approaches to paralysis and other motor function impairments.

In a study on rodents, the UC Irvine, UC San Diego and Harvard University team achieved this breakthrough by turning back the developmental clock in a molecular pathway critical for the growth of corticospinal tract nerve connections.

They did this by deleting an enzyme called PTEN (a phosphatase and tensin homolog), which controls a molecular pathway called mTOR that is a key regulator of cell growth. PTEN activity is low early during development, allowing cell proliferation. PTEN then turns on when growth is completed, inhibiting mTOR and precluding any ability to regenerate.

Trying to find a way to restore early-developmental-stage cell growth in injured tissue, Zhigang He, a senior neurology researcher at Children's Hospital Boston and Harvard Medical School, first showed in a 2008 study that blocking PTEN in mice enabled the regeneration of connections from the eye to the brain after optic nerve damage.

Oswald Steward, Ph.D. of
UC Irvine.

He then collaborated with Oswald Steward of UCI and Binhai Zheng of UCSD to see if the same approach could promote nerve regeneration in injured spinal cord sites. Results of their study appear online in Nature Neuroscience.

"Until now, such robust nerve regeneration has been impossible in the spinal cord," said Steward, anatomy & neurobiology professor and director of the Reeve-Irvine Research Center at UCI.

"Paralysis and loss of function from spinal cord injury has been considered untreatable, but our discovery points the way toward a potential therapy to induce regeneration of nerve connections following spinal cord injury in people."

According to Christopher & Dana Reeve Foundation data, about 2 percent of Americans have some form of paralysis resulting from spinal cord injury, which is due primarily to the interruption of connections between the brain and spinal cord.

An injury the size of a grape can lead to complete loss of function below the level of injury. For example, an injury to the neck can cause paralysis of arms and legs, loss of ability to feel below the shoulders, inability to control the bladder and bowel, loss of sexual function, and secondary health risks including susceptibility to urinary tract infections, pressure sores and blood clots due to an inability to move the legs.

"These devastating consequences occur even though the spinal cord below the level of injury is intact," Steward noted.

"All these lost functions could be restored if we could find a way to regenerate the connections that were damaged."

He and his colleagues are now studying whether the PTEN-deletion treatment leads to actual restoration of motor function in mice with spinal cord injury. Further research will explore the optimal timeframe and drug-delivery system for the therapy.

Friday, 6 August 2010

Human embryonic stem (ES) cells and adult cells reprogrammed to an embryonic stem cell-like state — so-called induced pluripotent stem or iPS cells — exhibit very few differences in their gene expression signatures and are nearly indistinguishable in their chromatin state, according to Whitehead Institute researchers.

The pluripotency of ES cells fuelled excitement over their use in regenerative medicine. While ethical hurdles associated with the clinical application of human ES cells appeared to have been overcome with the development of methods to create iPS cells, some recent research has suggested that ES and iPS cells have substantial differences in which sets of genes they express. These findings from Whitehead Institute argue to the contrary, rekindling hopes that, under the proper circumstances, iPS cells may indeed hold the clinical promise ascribed to them earlier.

Their results are published in the August 6 issue of Cell Stem Cell.

iPS cells are made by introducing three key genes into adult cells. These reprogramming factors push the cells from a mature state to a more flexible embryonic stem cell-like state. Like ES cells, iPS cells can then, in theory, be coaxed to mature into almost any type of cell in the body. Unlike ES cells, iPS cells taken from a patient are not likely to be rejected by that patient's immune system. This difference overcomes a major hurdle in regenerative medicine.

"Billions of dollars have been invested in the idea that we will use ES cells at some point in the future as therapeutic or regenerative agents, but for ethical and practical issues, this may not be possible," says Garrett Frampton, a co-first author on the Cell Stem Cell paper and a graduate student in the lab of Whitehead Member Richard Young.

"But if they work out therapies with ES cells, and iPS cells are equivalent to ES cells, then the idea is that those therapies could be used with iPS cells as well. Whereas if iPS cells are different from ES cells, then who knows if you can use iPS cells for therapy?"

Since iPS cells were first developed in 2006, the similarities and differences between ES and iPS cells have been hotly debated in the scientific community. Thus far, researchers have gauged the cells' equivalence by determining whether the cells express the same genes, but such studies have yielded mixed results.

In revisiting the question of the cells' equivalence, Frampton and co-first author Matthew Guenther, who is a scientist in the Young lab, analyzed gene expression patterns and the cells' chromatin structure. Chromatin is the packaging of DNA around a protein scaffold. Variations in chromatin "packaging" can themselves alter gene expression, yet Guenther and Frampton found that human iPS and ES cells to be almost identical in both gene expression and chromatin structure.

"At this stage, we can't yet prove that they are absolutely identical, but the available technology doesn't reveal differences," says Young, who is also a biology professor at MIT.

"It does mean that iPS cells could be useful as personal ES cells in the future."

Some earlier studies have indicated that iPS and ES cells are dissimilar enough to be classified as different cell types. To see why the results differed so strikingly from theirs, Guenther and Frampton reanalyzed those studies' data. They concluded that the differences noted in other studies were not consistent between different laboratories and thus were not likely to be a result of fundamental differences between the cell types.

"The key question is, are any of these differences functionally relevant? Do they change how a cell matures or not?" says Whitehead Member Rudolf Jaenisch, whose lab worked closely with Guenther and Frampton.

"The earlier documented differences were more noise than anything. But other tests may give you a different answer. So it is still an open question, something that the field will continue to struggle with and have to decide."

Guenther agrees.

"Our paper addresses the ground state of iPS and ES cells in a laboratory setting," he says.

"But we don't know for a fact that they won't behave differently when they mature into various cell types or tissues. That's the next step."

Scientists at the Gladstone Institute of Cardiovascular Disease (GICD) have found a new way to make beating heart cells from the body's own cells that could help regenerate damaged hearts. Over 5 million Americans suffer from heart failure because the heart has virtually no ability to repair itself after a heart attack. Only 2,000 hearts become available for heart transplant annually in the United States, leaving limited therapeutic options for the remaining millions. In research published in the current issue of Cell, scientists in the laboratory of GICD director Deepak Srivastava, MD, directly reprogrammed structural cells called fibroblasts in the heart to become beating heart cells called cardiomyocytes. In doing so, they also found the first evidence that unrelated adult cells can be reprogrammed from one cell type to another without having to go all the way back to a stem cell state.

The researchers, led by Masaki Ieda, MD, PhD, started off with 14 genetic factors important for formation of the heart and found that together they could reprogram fibroblasts into cardiomyocyte-like cells. Remarkably, a combination of just three of the factors (Gata4, Mef2c, and Tbx5) was enough to efficiently convert fibroblasts into cells that could beat like cardiomyocytes and turned on most of the same genes expressed in cardiomyocytes. When transplanted into mouse hearts 1 day after the three factors were introduced, fibroblasts turned into cardiomyocyte-like cells within the beating heart.

"Scientists have tried for 20 years to convert non-muscle cells into heart muscle, but it turns out we just needed the right combination of genes at the right dose," said Dr. Ieda.

"The ability to reprogram fibroblasts into cardiomyocytes has many therapeutic implications," explained Dr. Srivastava, senior author on the paper.

"Half of the cells in the heart are fibroblasts, so the ability to call upon this reservoir of cells already in the organ to become beating heart cells has tremendous promise for cardiac regeneration. Introducing the defined factors, or factors that mimic their effect, directly into the heart to create new heart muscle would avoid the need to inject stem cells into the heart and all the obstacles that go along with such cell-based therapies."

The study results imply that cells in multiple organs within an individual might be directed into necessary cell types to repair defects within the body.

This next generation of direct reprogramming builds on the reprogramming method discovered by Gladstone investigator Shinya Yamanaka, MD, PhD, who found that, by using four genetic factors, adult cells can be reprogrammed to pluripotent cells known as induced pluripotent stem (iPS) cells. Like embryonic stem cells, iPS cells can turn into any of the cell types of the human body.

However, direct cellular reprogramming that does not involve a stem cell state solves some of the safety concerns surrounding the use of stem cells. Going directly from one adult cell type to another would eliminate the risk that some stem cells might develop inappropriately to form tumours.

While direct reprogramming may offer some advantages over Yamanaka's original method, additional work will be necessary to refine the method and bring it closer to a practical therapeutic strategy.

"Direct reprogramming has not yet been done in human cells," Dr. Srivastava explained.

"And, the hope is still to find small molecules, rather than genetic factors, that can be used to direct the cell-fate switch."

Tissue regeneration a la salamanders and newts seems like it should be the stuff of science fiction. But it happens routinely. Why can't we mammals just re-grow a limb or churn out a few new heart muscle cells as needed? New research suggests there might be a very good reason: Restricting our cells' ability to pop in and out of the cell cycle at will — a prerequisite for the cell division necessary to make new tissue — reduces the chances that they'll run amok and form potentially deadly cancers.

Now scientists at the Stanford University School of Medicine have taken a big step toward being able to confer this regenerative capacity on mammalian muscle cells; they accomplished this feat in experiments with laboratory mice in which they blocked the expression of just two tumour-suppressing proteins. The finding may move us closer to future regenerative therapies in humans — surprisingly, by sending us shimmying back down the evolutionary tree.

"In contrast, mammals are pathetic. We can regenerate our livers, and that's about it. Until now it's been a mystery as to how they do it."

Blau is the senior author of the research, which will be published in Cell Stem Cell on Aug. 6. Kostandin Pajcini, PhD, a former graduate student, and Jason Pomerantz, MD, a former postdoctoral scholar in Blau's laboratory, are primarily responsible for the work and are first author and co-senior author, respectively.

Although there has been a lot of discussion about using adult or embryonic stem cells to repair or revitalize tissues throughout the body, in this case the researchers were not studying stem cells. Instead, they were investigating whether myocytes, run-of-the mill muscle cells that normally do not divide, can be induced to re-enter the cell cycle and begin proliferating. This is important because most specialized, or differentiated, cells in mammals are locked into a steady state that does not allow cell division. And without cell division, it is not possible to get regeneration.

In contrast, the cells of some types of amphibians are able to replace lost or damaged tissue by entering the cell cycle to give rise to more muscle cells. While doing so, the cells maintain their muscle identity, which prevents them from straying from the beaten path and becoming other, less useful cell types.

Pomerantz and Blau wondered if it could be possible to coax mammalian cells to follow a similar path. To do so, though, they needed to pinpoint what was different between mammalian and salamander cells when it comes to cell cycle control. One aspect involves a class of proteins called tumour suppressors that block inappropriate cell division.

Previous research had shown that a tumour suppressor called retinoblastoma, or Rb, plays an important role in preventing many types of specialized mammalian cells, including those found in muscle, from dividing willy-nilly. However, the effect of blocking the expression of Rb in mammalian cells has been inconsistent: In some cases it has allowed the cells to hop back into the cell cycle; in others, it hasn't.

The researchers employed some evolutionary detective work to figure out that another tumour suppressor called ARF might be involved. Like Rb, ARF works to throw the brakes on the cell cycle in response to internal signals. An examination of the evolutionary tree provided a key clue. They saw that ARF first arose in chickens. It is found in other birds and mammals, but not in animals like salamanders nestled on the lower branches. Tellingly, it's also missing in cell lines that begin cycling when Rb is lost, and it is expressed at lower-than-normal levels in mammalian livers — the only organ that we humans can regenerate.

Based on previous investigators' work with newts, Blau said it "seemed to us that they don't have the same limitations on growth. We hypothesized that maybe, during evolution, humans gained a tumour suppressor not present in lower animals at the expense of regeneration."

Sure enough, Pajcini and Pomerantz found that blocking the expression of both Rb and ARF allowed individual myocytes isolated from mouse muscle to dedifferentiate and begin dividing. When they put the cells back into the mice, they were able to merge with existing muscle fibres — as long as Rb expression was restored. Without Rb the transplanted cells proliferated excessively and disrupted the structure of the original muscle.

"These myocytes have reached the point of no return," said Blau.

"They can't just start dividing again. But here we show that temporarily blocking the expression of just two proteins can restore an ancient ability to contribute to mammalian muscle."

The key word here is "temporarily." As is clear from the mouse experiments, blocking the expression of tumour suppressors in mammalian cells can be a tricky gambit. Permanently removing these proteins can lead to uncontrolled cell division. But, a temporary and well-controlled loss — as the researchers devised here — could be a useful therapeutic tool.

The research required some sophisticated technology to separate individual myocytes from one another for study. To do so, Pajcini travelled to Munich to learn how to optimize a technique normally used on cryopreserved and fixed tissue sections — "laser micro-dissection catapulting" — for use with living cells. But the effort paid off when he was able to prove conclusively that once the expression of the two proteins was blocked, individual live cells were, in fact, dividing in culture.

Next, the researchers would like to see if the technique works in other cell types, like those of the pancreas or the heart, and whether they can induce it to happen in tissue at sites of injury. If so, it may be possible to trigger temporary cell proliferation as a means of therapy for a variety of ailments.